Nicotinamide riboside Induced Energy Stress and Metabolic Reprogramming in BEAS-2B Cells

Nicotinamide riboside (NR), a NAD+ precursor, has received attention due to several health benefits it has induced in experimental models. Studies in cultured cells, animals, and humans consistently show increased NAD+ availability after NR supplementation, which is considered the only mode of NR action that leads to health benefits. In the present study, we show that a persistently low NR concentration (1 μM) in the growth medium of BEAS-2B human cells, grown in a monolayer, induces energy stress, which precedes a cellular NAD+ increase after 192 h. NR concentrations greater than 1 μM under the specified conditions were cytotoxic in the 2D cell culture model, while all concentrations tested in the 3D cell culture model (BEAS-2B cell spheroids exposed to 1, 5, 10, and 50 μM NR) induced apoptosis. Shotgun proteomics revealed that NR modulated the abundance of proteins, agreeing with the observed effects on cellular energy metabolism and cell growth or survival. Energy stress may activate pathways that lead to health benefits such as cancer prevention. Accordingly, the premalignant 1198 cell line was more sensitive to NR cytotoxicity than the phenotypically normal parent BEAS-2B cell line. The role of a mild energy stress induced by low concentrations of NR in its beneficial effects deserves further investigation. On the other hand, strategies to increase the bioavailability of NR require attention to toxic effects that may arise.


Table of Contents
Table S1 page S3  Table S1.ATP, ADP and AMP levels (pmol/µg protein) in BEAS-2B cells exposed or not to nicotinamide riboside (NR) from 24 to 192 h.
Asterisks indicate the significant difference between the NR exposed and control cells in each time point (p < 0.05).Unpaired t test with Welch's correction was used for the analyses.Table S4.Nineteen proteins associated to 46 representative terms and pathways selected among the significant terms and pathways of Table S3 and Figure S4).Selection criteria: at least 2 genes from the loaded list associated with a term (Number Genes) and representing at least 5% of the total number of genes in the term (% Associated Genes).The proteins belong to the cluster of 42 more abundant proteins in the NR group.Table S6.Twelve proteins associated to 36 representative terms and pathways selected among the significant terms and pathways of Table S5 and

Figure S5
). Selection criteria: at least 2 genes from the loaded list associated with a term (Number Genes) and representing at least 5% of the total number of genes in the term (% Associated Genes).The proteins belong to the cluster of 35 less abundant proteins in the NR group.It catalyzes the post-translational formation of 4hydroxyproline in -Xaa-Pro-Gly-sequences in collagens and other proteins, which is essential to the proper threedimensional folding of procollagen chains.
It was verified that P4HA2 is highly expressed in lung adenocarcinoma tumor cells.High P4HA2 expression indicated a poor prognosis and served as an independent prognostic risk factor in lung cancer f .Reaction: type I collagen synthesis in the context of osteogenesis imperfecta; protein hydroxylation; peptidyl-proline hydroxylation; peptidylproline hydroxylation to 4-hydroxy-L-proline; peptidyl-proline dioxygenase activity; procollagen-proline dioxygenase activity; peptidyl-proline 4-dioxygenase activity SERPINH1 serpin H1 (HSP47) A member of the serpin superfamily of serine proteinase inhibitors.It binds specifically to collagen and has a role as a chaperone in the biosynthetic pathway of collagen.
Its expression is associated with cancer growth and metastasis in several types of cancers.The presence of a high number of HSP47-positive fibroblasts in the cancer type I collagen synthesis in the context of osteogenesis imperfecta stroma was a risk factor for recurrence of lung cancer after surgery g .

PSAT1 phosphoserine aminotransferase
It is a phosphoserine aminotransferase member of the class-V pyridoxal-phosphate-dependent aminotransferase family.Catalyzes the transamination of 3phosphonooxypyruvate and L-glutamate to Ophosphoserine and 2-oxoglutarate in the pathway of Lserine biosynthesis.
It is overexpressed in tumors.Knockdown of PSAT1 suppressed cell proliferation, migration, and invasion of non-small cell lung cancer (NSCLC) h .

Reaction:
O-phospho-L-serine + 2-oxoglutarate <=> 3phosphonooxypyruvate + L-glutamate serine biosynthesis; serine metabolism; L-serine metabolic process; serine family amino acid biosynthetic process; L-serine biosynthetic process It is a thiol protease that belongs to the ubiquitin Cterminal hydrolase family of deubiquitinases.It hydrolyzes a peptide bond at the C-terminal glycine of ubiquitin and eliminates ubiquitin isopeptide from target proteins.This process prevents protein degradation by the ubiquitin-proteasome system and is important for regulation of protein turnover in cells.
UCHL1 was shown to be up regulated in urothelial bladder carcinoma cells k , and highly associated with the recurrence and invasion in breast cancer patients Figure S1page S23 -S24 belongs to a subfamily of the phosphotransferases.Catalyzes the last irreversible step in the biosynthesis of L-serine.It is overexpressed in cancer, e.g.non-small cell lung cancer (NSCLC).Decreased expression of PSPH inhibited NSCLC cell migration, invasion, and proliferation i .Reaction: serine biosynthesis; serine metabolism; L-serine metabolic process; serine family amino acid biosynthetic process; L-serine biosynthetic process O-phospho-L-serine + H 2 O => L-serine + response to ionizing radiation; double-strand break repair; and positive regulation of protein phosphorylation.It is overexpressed in cancer and correlated with poor overall survival in lung cancer patients.Its depletion inhibited cell epithelial-mesenchymal transition (EMT), proliferation, colony formation, migration, and invasion j .TP53 regulates transcription of additional cell cycle genes whose exact role in the p53 pathway remain uncertain

Table S8 .
Precision of the analytical methods for quantification of NAD + , ATP, ADP, AMP, and lactate in BEAS-2B cells by HPLC-ESI-MS/MS.